Structural Insights into the TLA-3 Extended-Spectrum ß-Lactamase and Its Inhibition by Avibactam and OP0595.

The development of effective inhibitors that block extended-spectrum ß-lactamases (ESBLs) and restore the action of ß-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants (Kiapp) of 1.71 ± 0.10 and 1.49 ± 0.05 µM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing Escherichia coli strain. The value of the second-order acylation rate constant (k2/K, where k2 is the acylation rate constant and K is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 103 M-1 · s-1] was closer to that of class C and D ß-lactamases (k2/K, <104 M-1 · s-1) than that of class A ß-lactamases (k2/K, >104 M-1 · s-1). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the ß5 and ß6 strands (insertion after Ser237), which appear only in PER-type class A ß-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency (kcat/Km ) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae.

In Vitro and In Vivo Activities of OP0595, a New Diazabicyclooctane, against CTX-M-15-Positive Escherichia coli and KPC-Positive Klebsiella pneumoniae.

Gram-negative bacteria are evolving to produce ß-lactamases of increasing diversity that challenge antimicrobial chemotherapy. OP0595 is a new diazabicyclooctane serine ß-lactamase inhibitor which acts also as an antibiotic and as a ß-lactamase-independent ß-lactam "enhancer" against Enterobacteriaceae Here we determined the optimal concentration of OP0595 in combination with piperacillin, cefepime, and meropenem, in addition to the antibacterial activity of OP0595 alone and in combination with cefepime, in in vitro time-kill studies and an in vivo infection model against five strains of CTX-M-15-positive Escherichia coli and five strains of KPC-positive Klebsiella pneumoniae An OP0595 concentration of 4 µg/ml was found to be sufficient for an effective combination with all three ß-lactam agents. In both in vitro time-kill studies and an in vivo model of infection, cefepime-OP0595 showed stronger efficacy than cefepime alone against all ß-lactamase-positive strains tested, whereas OP0595 alone showed weaker or no efficacy. Taken together, these data indicate that combinational use of OP0595 and a ß-lactam agent is important to exert the antimicrobial functions of OP0595.

OP0595, a new diazabicyclooctane: mode of action as a serine ß-lactamase inhibitor, antibiotic and ß-lactam 'enhancer'.

OBJECTIVES: The production of a growing diversity of ß-lactamases by Gram-negative bacteria challenges antimicrobial chemotherapy. OP0595, discovered separately by each of Meiji Seika Pharma and Fedora Pharmaceuticals, is a new diazabicyclooctane serine ß-lactamase inhibitor that also acts as an antibiotic and as a ß-lactamase-independent ß-lactam 'enhancer'. METHODS: Inhibitory activity against serine ß-lactamases and affinity for PBPs were determined using nitrocefin and Bocillin FL, respectively. MICs alone and in combination with ß-lactam agents were measured according to CLSI recommendations. Morphological changes in Escherichia coli were examined by phase-contrast microscopy. RESULTS: IC50s of OP0595 for class A and C ß-lactamases were <1000 nM, with covalent binding demonstrated to the active-site serine of CTX-M-44 and AmpC enzymes. OP0595 also had direct antibiotic activity against many Enterobacteriaceae, associated with inhibition of PBP2 and conversion of the bacteria into spherical forms. Synergy between OP0595 and ß-lactam agents was seen against strains producing class A and C ß-lactamases vulnerable to inhibition. Lastly, OP0595 lowered the MICs of PBP3-targeted partner ß-lactam agents for a non-ß-lactamase-producing E. coli mutant that was resistant to OP0595 itself, indicating ß-lactamase-independent 'enhancer'-based synergy. CONCLUSIONS: OP0595 acts in three ways: (i) as an inhibitor of class A and C ß-lactamases, covalently binding at their active sites; (ii) as an antibacterial, by inhibiting PBP2 of several Enterobacteriaceae; and (iii) as an 'enhancer' of ß-lactam agents that bind to other PBPs besides PBP2 for several Enterobacteriaceae. OP0595 has considerable potential to overcome resistance when it is combined with various ß-lactam agents.

X-ray crystallographic analysis of IMP-1 metallo-ß-lactamase complexed with a 3-aminophthalic acid derivative, structure-based drug design, and synthesis of 3,6-disubstituted phthalic acid derivative inhibitors.

3-(4-Hydroxypiperidine-1-yl) phthalic acid 1 shows potent inhibitory activity against metallo-ß-lactamase, which is known to inactivate ß-lactam antibiotics such as carbapenems. Here, the structure of co-crystals of the metallo-ß-lactamase IMP-1 and 1 was first analyzed by X-ray crystallography, and then used for structure-based drug design. Four novel compounds bearing substituents at the 6-position were synthesized to produce 3,6-disubstituted phthalic acid derivatives, and their IMP-1 inhibitory activity and synergistic effect with the carbapenem biapenem (BIPM) were evaluated. 3,6-Disubstituted phthalic acid derivatives showed potent IMP-1 inhibitory activity. In particular, compound 13 showed 10-fold higher IMP-1 inhibitory activity as compared with the parent derivative 1.

Activity of carbapenems with ME1071 (disodium 2,3-diethylmaleate) against Enterobacteriaceae and Acinetobacter spp. with carbapenemases, including NDM enzymes.

OBJECTIVES: ME1071 is a maleic acid that inhibits metallo-ß-lactamases (MBLs). We examined its ability to potentiate different carbapenems against MBL-producing Enterobacteriaceae in relation to its inhibition kinetics. METHODS: Enterobacteriaceae and Acinetobacter isolates with IMP, VIM and NDM MBLs were tested; bacteria with other types of carbapenem resistance were used as controls. Chequerboard titrations were performed by CLSI agar dilution, carbapenemases were cloned into pET-28a(+) and purified by column chromatography, and kinetic parameters were determined by spectrophotometry. RESULTS: The key findings were: (i) the MICs of carbapenems varied widely among isolates with the same carbapenemase, but those with the NDM types were generally the most resistant; (ii) biapenem was the carbapenem least compromised by all MBL types, owing to weaker kinetic efficiency (k(cat)/K(m)) for hydrolysis, contingent on lower affinity (higher K(m)); (iii) MBLs were the only carbapenemases inhibited by ME1071, confirming its specificity of action; and (iv) irrespective of the partner carbapenem, synergy with ME1071 was least for organisms with NDM MBLs and most for those with IMP types, correlating with ME1071 having weakest affinity (highest K(i)) for NDM-1 and strongest affinity for IMP-1. CONCLUSIONS: ME1071 reduced the MICs of carbapenems for bacteria with NDM-1 enzyme though synergy was weaker than for bacteria with IMP and VIM metallo-enzymes; this correlated with ME1071 having weaker affinity for NDM-1 than IMP-1 and VIM-2. As the weakest MBL substrate carbapenem, biapenem was the easiest to protect.

Metallo-ß-lactamase inhibitory activity of 3-alkyloxy and 3-amino phthalic acid derivatives and their combination effect with carbapenem.

3-Alkyloxy and 3-amino phthalic acid derivatives were found to have metallo-ß-lactamase inhibitory activity. Among them, 3-amino phthalic acid derivatives showed both potent activity against metallo-ß-lactamase, IMP-1 inhibitory activity and a strong combination effect with biapenem (BIPM), carbapenem antibiotic. In particular, the 4'-hydroxy-piperidine derivative showed strong IMP-1 inhibitory activity and a combination effect with various antibiotics.

Metallo-beta-lactamase inhibitory activity of phthalic acid derivatives.

4-Butyl-3-methylphthalic acid was recognized as a metallo-beta-lactamase inhibitor. The structure-activity relationship study of substituted phthalic acids afforded 3-phenylphthalic acid derivatives as potent IMP-1 inhibitors. On the other hand, 3-substituted with 4-hydroxyphenyl phthalic acid derivative displayed a potent combination effect with biapenem (BIPM) against Pseudomonas aeruginosa that produce IMP-1.

In vitro and in vivo activities of the diazabicyclooctane OP0595 against AmpC-derepressed Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common cause for healthcare-associated infections, which have been historically treated by antipseudomonal ß-lactam agents in the clinical setting. However, P. aeruginosa has evolved to overcome these ß-lactam agents via multiple endogenous resistance mechanisms, including derepression of the chromosomal cephalosporinase (AmpC). In this article, we investigated the effective concentration of OP0595 for combination with piperacillin, cefepime or meropenem in in vitro susceptibility tests, and the antibacterial activity of cefepime in combination with OP0595 in both in vitro time-kill studies and in vivo murine thigh infection model study with AmpC-derepressed P. aeruginosa. The sufficient combinational concentration of OP0595 was a 4 µg ml-1 with all these three ß-lactam agents. OP0595 increased the antibacterial activity of cefepime in both in vitro and in vivo studies against all strains tested. Taken together, OP0595 is the diazabicyclooctane serine ß-lactamase inhibitor with activity against AmpC-derepressed P. aeruginosa and its combinational use with a ß-lactam agent will provide a new approach for the treatment of P. aeruginosa infections.